The Cavin-1/Caveolin-1 interaction attenuates BMP/Smad signaling in pulmonary hypertension by interfering with BMPR2/Caveolin-1 binding

Caveolin-1 (CAV1) and Cavin-1 are components of caveolae, both of which interact with and influence the composition and stabilization of caveolae. CAV1 is associated with pulmonary arterial hypertension (PAH). Bone morphogenetic protein (BMP) type 2 receptor (BMPR2) is localized in caveolae associated with CAV1 and is commonly mutated in PAH. Here, we show that BMP/Smad signaling is suppressed in pulmonary microvascular endothelial cells of CAV1 knockout mice. Moreover, hypoxia enhances the CAV1/Cavin-1 interaction but attenuates the CAV1/BMPR2 interaction and BMPR2 membrane localization in pulmonary artery endothelial cells (PAECs). Both Cavin-1 and BMPR2 are associated with the CAV1 scaffolding domain. Cavin-1 decreases BMPR2 membrane localization by inhibiting the interaction of BMPR2 with CAV1 and reduces Smad signal transduction in PAECs. Furthermore, Cavin-1 knockdown is resistant to CAV1-induced pulmonary hypertension in vivo. We demonstrate that the Cavin-1/Caveolin-1 interaction attenuates BMP/Smad signaling and is a promising target for the treatment of PAH.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.

A description of all covariates tested
A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g.means) or other basic estimates (e.g.regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g.confidence intervals) For null hypothesis testing, the test statistic (e.g.F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g.Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection Fluorescent signals were detected using a Keyence BZ-X700 digital microscope (Osaka, Japan) or Zeiss LSM510 META Confocal Imaging System (Oberkochen, Germany).PLA signal was captured using a Keyence BZ-X700 digital microscope.For the transmission electron microscopy, microtome sections were examined under an H-7100 transmission electron microscope (HITACHI, Tokyo, Japan).luminescence in apoptosis assay was measured using a TECAN microplate reader (Zurich, Switzerland).To measure RV hemodynamics, open-chest RV catheterization using a 1.2-F pressure catheter (Transonic Scisense, Inc., London, ON, Canada) was performed.LC-MS/MS analyses were performed using a nano LC (UltiMate® 3000) (Dionex, Sunnyvale, CA, USA) coupled with a Q Exactive Plus Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA).Instrument operation and data acquisition were performed using Xcalibur Software (Thermo Scientific, Waltham, MA, USA).

Data analysis
The MS/MS raw data were processed using Mascot version 2.6.0 (Matrix Sciences, London, US) and were searched in the Swiss-Prot database with humans as the species, carbamidomethylation of cysteine as a static modification, oxidation of methionine as a dynamic modification, precursor mass tolerance of 1.0 Da, and a fragment mass tolerance of 0.8 Da.The dat files of all fractions obtained were processed with Scaffold version 5.0.1 (Proteome Software Inc.).All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, Inc., CA, USA).
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers.We strongly encourage code deposition in a community repository (e.g.GitHub).See the Nature Portfolio guidelines for submitting code & software for further information.